Developing a competitive hematopoietic repopulating assay in zebrafish Project Summary/Abstract Hematopoietic stem cells (HSCs) precisely regulate the balance between self-renewal and differentiation to generate appropriate numbers of mature blood cells required by an organism. When genes that direct these decisions are dysregulated, unchecked self-renewal can result in hematopoietic malignancy. Characterizing the decision-making processes regulating HSC function is critically important to understand leukemogenesis and also to enhance HSC transplantation strategies used to treat many human cancers and blood diseases. My laboratory is capitalizing on the zebrafish animal model to uncover new genetic regulators of HSCs. I have developed an immune- matched transgenic zebrafish model to phenocopy murine models of competitive repopulation using clonal CG2 zebrafish. To compare the relative engraftment potential of mutant zebrafish HSCs with wild type HSCs, transgenic CG2:ubiquitin-GFP and CG2:ubiquitin-DsRedE2 donor fish with fluorochrome positive marrow cells are transplanted into fluorochrome negative clonal recipients, resulting in long term, multi-lineage engraftment. A preliminary pilot screen of zebrafish mutants with abnormal embryonic HSC development identified two haploinsufficient mutants with impaired recovery of hematopoietic precursor cells in the marrow after sublethal radiation injury. Both phospholipase C gamma1 (plc?1) and histone deacetylase 1 (hdac1) have previously been identified as playing a role in self-renewal, and our data suggest they may be important for regulating HSC homeostasis. I will characterize and refine our competitive hematopoietic repopulating assay by testing the hypothesis that PLC?1 and HDAC1 haploinsufficiency will result in impaired hematopoietic engraftment. In addition, using random insertional mutagenesis of clonal zebrafish donors, I will perform an unbiased forward mutagenesis screen to identify genes that alter HSC engraftment. Marrow from heterozygous and/or homozygous mutant adults will be used in my competitive hematopoietic repopulating assay to identify those mutants with skewed marrow repopulation favoring one of the competitive donors. I am confident this assay will identify known and unknown genes important for multiple aspects of hematopoietic engraftment. This work will generate mutant zebrafish to use as tools to dissect the complex regulatory network that determines HSC function. Characterizing these genes will accelerate our understanding of normal HSC function in vivo and after transplantation, as well as providing new insights into the mechanisms that cause leukemia.